Simian Immunodeficiency Virus Sequences Research Articles

The Landscape of Persistent Viral Genomes in ART-Treated SIV, SHIV, and HIV-2 Infections

Evaluation of HIV cure strategies is complicated by defective proviruses that persist in ART-treated patients but are irrelevant to cure. Non-human primates (NHP) are essential for testing cure strategies.However, the persisting proviral landscape in ART-treated NHPs is uncharacterized. Here, we describe viral genomes persisting in ART-treated, simian immunodeficiency virus (SIV)-infected NHPs, simian-human immunodeficiency virus (SHIV)-infected NHPs, and humans infected with HIV-2, an SIV-related virus. The landscapes of persisting SIV, SHIV, and HIV-2 genomes are also dominated by defective sequences. However, there was a significantly higher fraction of intact SIV proviral genomes compared to ART-treated HIV-1 or HIV-2 infected humans. Compared to humans with HIV-1, SIV-infected NHPs had more hypermutated genomes, a relative paucity of clonal SIV sequences, and a lower frequency of deleted genomes. Finally, we report an assay for measuring intact SIV genomes which may have value in cure research.

Rare Control of SIVmac239 Infection in a Vaccinated Rhesus Macaque.

Effector memory T cell (TEM) responses display potent antiviral properties and have been linked to stringent control of simian immunodeficiency virus (SIV) replication. Since recurrent antigen stimulation drives the differentiation of CD8+ T cells toward the TEM phenotype, in this study we incorporated a persistent herpesviral vector into a heterologous prime/boost/boost vaccine approach to maximize the induction of TEM responses. This new regimen resulted in CD8+ TEM-biased responses in four rhesus macaques, three of which controlled viral replication to <1,000 viral RNA copies/ml of plasma for more than 6 months after infection with SIVmac239. Over the course of this study, we made a series of interesting observations in one of these successful controller animals. Indeed, in vivo elimination of CD8αβ+ T cells using a new CD8β-depleting antibody did not abrogate virologic control in this monkey. Only after its CD8α+ lymphocytes were depleted did SIV rebound, suggesting that CD8αα+ but not CD8αβ+ cells were controlling viral replication. By 2 weeks postinfection (PI), the only SIV sequences that could be detected in this animal harbored a small in-frame deletion in nef affecting six amino acids. Deep sequencing of the SIVmac239 challenge stock revealed no evidence of this polymorphism. However, sequencing of the rebound virus following CD8α depletion at week 38.4 PI again revealed only the six-amino acid deletion in nef. While any role for immunological pressure on the selection of this deleted variant remains uncertain, our data provide anecdotal evidence that control of SIV replication can be maintained without an intact CD8αβ+ T cell compartment.

Linking pig-tailed macaque major histocompatibility complex class I haplotypes and cytotoxic T lymphocyte escape mutations in simian immunodeficiency virus infection.

The influence of major histocompatibility complex class I (MHC-I) alleles on human immunodeficiency virus (HIV) diversity in humans has been well characterized at the population level. MHC-I alleles likely affect viral diversity in the simian immunodeficiency virus (SIV)-infected pig-tailed macaque (Macaca nemestrina) model, but this is poorly characterized. We studied the evolution of SIV in pig-tailed macaques with a range of MHC-I haplotypes. SIV(mac251) genomes were amplified from the plasma of 44 pig-tailed macaques infected with SIV(mac251) at 4 to 10 months after infection and characterized by Illumina deep sequencing. MHC-I typing was performed on cellular RNA using Roche/454 pyrosequencing. MHC-I haplotypes and viral sequence polymorphisms at both individual mutations and groups of mutations spanning 10-amino-acid segments were linked using in-house bioinformatics pipelines, since cytotoxic T lymphocyte (CTL) escape can occur at different amino acids within the same epitope in different animals. The approach successfully identified 6 known CTL escape mutations within 3 Mane-A1*084-restricted epitopes. The approach also identified over 70 new SIV polymorphisms linked to a variety of MHC-I haplotypes. Using functional CD8 T cell assays, we confirmed that one of these associations, a Mane-B028 haplotype-linked mutation in Nef, corresponded to a CTL epitope. We also identified mutations associated with the Mane-B017 haplotype that were previously described to be CTL epitopes restricted by Mamu-B*017:01 in rhesus macaques. This detailed study of pig-tailed macaque MHC-I genetics and SIV polymorphisms will enable a refined level of analysis for future vaccine design and strategies for treatment of HIV infection. Cytotoxic T lymphocytes select for virus escape mutants of HIV and SIV, and this limits the effectiveness of vaccines and immunotherapies against these viruses. Patterns of immune escape variants are similar in HIV type 1-infected human subjects that share the same MHC-I genes, but this has not been studied for SIV infection of macaques. By studying SIV sequence diversity in 44 MHC-typed SIV-infected pigtail macaques, we defined over 70 sites within SIV where mutations were common in macaques sharing particular MHC-I genes. Further, pigtail macaques sharing nearly identical MHC-I genes with rhesus macaques responded to the same CTL epitope and forced immune escape. This allows many reagents developed to study rhesus macaques to also be used to study pigtail macaques. Overall, our study defines sites of immune escape in SIV in pigtailed macaques, and this enables a more refined level of analysis of future vaccine design and strategies for treatment of HIV infection.

Genital Tract Sequestration of SIV following Acute Infection

We characterized the evolution of simian immunodeficiency virus (SIV) in the male genital tract by examining blood- and semen-associated virus from experimentally and sham vaccinated rhesus monkeys during primary infection. At the time of peak virus replication, SIV sequences were intermixed between the blood and semen supporting a scenario of high-level virus “spillover” into the male genital tract. However, at the time of virus set point, compartmentalization was apparent in 4 of 7 evaluated monkeys, likely as a consequence of restricted virus gene flow between anatomic compartments after the resolution of primary viremia. These findings suggest that SIV replication in the male genital tract evolves to compartmentalization after peak viremia resolves.

Dating the Age of the SIV Lineages That Gave Rise to HIV-1 and HIV-2

Great strides have been made in understanding the evolutionary history of simian immunodeficiency virus (SIV) and the zoonoses that gave rise to HIV-1 and HIV-2. What remains unknown is how long these SIVs had been circulating in non-human primates before the transmissions to humans. Here, we use relaxed molecular clock dating techniques to estimate the time of most recent common ancestor for the SIVs infecting chimpanzees and sooty mangabeys, the reservoirs of HIV-1 and HIV-2, respectively. The date of the most recent common ancestor of SIV in chimpanzees is estimated to be 1492 (1266–1685), and the date in sooty mangabeys is estimated to be 1809 (1729–1875). Notably, we demonstrate that SIV sequences sampled from sooty mangabeys possess sufficient clock-like signal to calibrate a molecular clock; despite the differences in host biology and viral dynamics, the rate of evolution of SIV in sooty mangabeys is indistinguishable from that of its human counterpart, HIV-2. We also estimate the ages of the HIV-2 human-to-human transmissible lineages and provide the first age estimate for HIV-1 group N at 1963 (1948–1977). Comparisons between the SIV most recent common ancestor dates and those of the HIV lineages suggest a difference on the order of only hundreds of years. Our results suggest either that SIV is a surprisingly young lentiviral lineage or that SIV and, perhaps, HIV dating estimates are seriously compromised by unaccounted-for biases.

Impaired RNA incorporation and dimerization in live attenuated leader-variants of SIVmac239

BackgroundThe 5' untranslated region (UTR) or leader sequence of simian immunodeficiency virus (SIVmac239) is multifunctional and harbors the regulatory elements for viral replication, persistence, gene translation, expression, and the packaging and dimerization of viral genomic RNA (vRNA). We have constructed a series of deletions in the SIVmac239 leader sequence in order to determine the involvement of this region in both the packaging and dimerization of viral genomic RNA. We also assessed the impact of these deletions upon viral infectiousness, replication kinetics and gene expression in cell lines and monkey peripheral blood mononuclear cells (PBMC).ResultsRegions on both sides of the major splice donor (SD) were found to be necessary for the efficiency and specificity of viral genome packaging. However, stem-loop1 is critical for both RNA encapsidation and dimerization. Downstream elements between the splice donor and the initiation site of SIV-Gag have additive effects on RNA packaging and contribute to a lesser degree to RNA dimerization. The targeted disruption of structures on both sides of the SD also severely impacts viral infectiousness, gene expression and replication in both CEMx174 cells and rhesus PBMC.ConclusionIn the leader region of SIVmac239, stem-loop1 functions as the primary determinant for both RNA encapsidation and dimerization. Downstream elements between the splice donor and the translational initiation site of SIV-Gag are classified as secondary determinants and play a role in dimerization. Collectively, these data signify a linkage between the primary encapsidation determinant of SIVmac239 and RNA dimerization.

Short Communication:A Rapid and Sensitive Real-Time PCR Assay for the K65R Drug Resistance Mutation in SIV Reverse Transcriptase

Macaques infected with simian immunodeficiency virus (SIV) provide a suitable model for assessing the efficacy of antiretroviral (ARV) drug interventions and drug resistance selection associated with treatment. Resistance to the HIV reverse transcriptase inhibitor tenofovir continues to be examined in different treatment strategies in the macaque model. Evaluations of treatment interventions and drug resistance are hampered by the limited sensitivity of conventional population sequencing and the substantial effort involved in testing various tissue compartments in which viruses may reside. Therefore, a sensitive assay that permits simple and rapid testing for drug-resistant viruses would benefit appraisals of ARV treatments using in vivo models. To have this capability, we developed a real-time PCR-based assay for the detection of the SIV K65R reverse transcriptase mutation, a key marker for reduced susceptibility to tenofovir. Evaluations of SIV sequences yielded an assay detection limit mean of 0.4% mutant virus (range = 0.1-2%) in a wild-type background. In testing longitudinal plasma specimens from four SIV-infected macaques that received an active daily regimen of 30 mg/kg of tenofovir subcutaneously, the assay was able to detect K65R-positive viruses in all animals within 1-7 weeks after treatment began. The emerging mutants were initially present at frequencies estimated between 0.4% and 3%, below the detection capability of population sequencing. We propose the SIV K65R real-time PCR assay provides improved sensitivity and simplicity in studying tenofovir resistance in macaque models.

A novel simian immunodeficiency virus from black mangabey (Lophocebus aterrimus) in the Democratic Republic of Congo

In order to understand primate lentivirus evolution, characterization of additional simian immunodeficiency virus (SIV) strains is essential. Here, an SIV from a black mangabey (Lophocebus aterrimus) originating from the Democratic Republic of Congo was analysed phylogenetically. The monkey had cross-reactive antibodies against human immunodeficiency virus type 1 (HIV-1) and HIV-2. The viral pol region sequence was amplified by nested PCR and sequence analysis confirmed that it was related to known SIV sequences. This is the first report to characterize genetically an SIV from the monkey genus Lophocebus. Phylogenetic analysis of the pol region revealed that this novel SIV, designated SIVbkm, fell into the SIVsyk and SIVgsn virus group, containing viruses isolated from the genus Cercopithecus, and suggests that cross-species transmission has occurred between species of the genera Lophocebus and Cercopithecus.

Humoral Immunity and the Evolution of HIV-2

Human immunodeficiency virus type 2 (HIV-2) evolved from the zoonotic transmission of simian immunodeficiency virus (SIV) that naturally infects sooty mangabeys found in West Africa. Using sera from HIV-2–infected humans, we discovered that an hypervariable region (the V4 loop) of HIV- 2 induces antibody responses only weakly reactive against itself but strongly reactive against analogous sequences from the V4 loop of strains of SIV. Available sequence data indicates that all strains of HIV-2 have large deletions in the V4 region that truncate an immunodominant neutralizing B cell epitope among strains of SIV. Infection of a macaque with a sequenced clone of HIV-2 similarly elicited antibodies that poorly recognized the V4 loop of HIV-2 but readily bound to analogous SIV sequences. Our data are consistent with a scenario whereby a disparate antibody response directed against the V4 loop may have influenced the selective expansion and survival of HIV-2 in humans.

Humoral immunity and the evolution of HIV-2.

Human immunodeficiency virus type 2 (HIV-2) evolved from the zoonotic transmission of simian immunodeficiency virus (SIV) that naturally infects sooty mangabeys found in West Africa. Using sera from HIV-2-infected humans, we discovered that an hypervariable region (the V4 loop) of HIV-2 induces antibody responses only weakly reactive against itself but strongly reactive against analogous sequences from the V4 loop of strains of SIV. Available sequence data indicates that all strains of HIV-2 have large deletions in the V4 region that truncate an immunodominant neutralizing B cell epitope among strains of SIV. Infection of a macaque with a sequenced clone of HIV-2 similarly elicited antibodies that poorly recognized the V4 loop of HIV-2 but readily bound to analogous SIV sequences. Our data are consistent with a scenario whereby a disparate antibody response directed against the V4 loop may have influenced the selective expansion and survival of HIV-2 in humans.

Promoting trimerization of soluble human immunodeficiency virus type 1 (HIV-1) Env through the use of HIV-1/simian immunodeficiency virus chimeras.

The envelope proteins (Env) of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) form homo-oligomers in the endoplasmic reticulum. The oligomeric structure of Env is maintained, but is less stable, after cleavage in a Golgi compartment and transport to the surface of infected cells. Functional, virion-associated HIV-1 and SIV Env have an almost exclusively trimeric structure. In addition, a soluble form of SIV Env (gp140) forms a nearly homogeneous population of trimers. Here, we describe the oligomeric structure of soluble, uncleaved HIV-1 gp140 and modifications that promote a stable trimeric structure. Biochemical and biophysical analyses, including sedimentation equilibrium and scanning transmission electron microscopy, revealed that unmodified HIV-1 gp140 purified as a heterogeneous range of oligomeric species, including dimers and aggregates. Deletion of the V2 domain alone or, especially, both the V1 and V2 domains reduced dimer formation but promoted aggregation rather than trimerization. Expressing gp140 with mannose-only oligosaccharides did not eliminate heterogeneity. Replacement of the entire gp41 segment of HIV-1 gp140 or just the N-terminal half (85 amino acids) of this segment with the corresponding region of SIV was sufficient to confer efficient trimerization for gp140 derived from clade B and C isolates. Importantly, the relatively small segment of the HIV Env replaced by SIV sequences contains no known targets of neutralizing antibody. The soluble trimeric form of HIV-1 Env should prove useful for assessment of antigenic structure and immunogenicity.

Simian-human immunodeficiency virus escape from cytotoxic T-lymphocyte recognition at a structurally constrained epitope.

Virus-specific cytotoxic T lymphocytes (CTL) exert intense selection pressure on replicating simian immunodeficiency virus (SIV) and human immunodeficiency virus type 1 (HIV-1) in infected individuals. The immunodominant Mamu-A(*)01-restricted Gag p11C, C-M epitope is highly conserved among all sequenced isolates of SIV and therefore likely is structurally constrained. The strategies used by virus isolates to mutate away from an immunodominant epitope-specific CTL response are not well defined. Here we demonstrate that the emergence of a position 2 p11C, C-M epitope substitution (T47I) in a simian-human immunodeficiency virus (SHIV) strain 89.6P-infected Mamu-A(*)01(+) monkey is temporally correlated with the emergence of a flanking isoleucine-to-valine substitution at position 71 (I71V) of the capsid protein. An analysis of the SIV and HIV-2 sequences from the Los Alamos HIV Sequence Database revealed a significant association between any position 2 p11C, C-M epitope mutation and the I71V mutation. The T47I mutation alone is associated with significant decreases in viral protein expression, infectivity, and replication, and these deficiencies are restored to wild-type levels with the introduction of the flanking I71V mutation. Together, these data suggest that a compensatory mutation is selected for in SHIV strain 89.6P to facilitate the escape of that virus from CTL recognition of the dominant p11C, C-M epitope.

Analysis of the immune response and viral evolution during the acute phase of SIV infection

Development of an effective vaccine against human immunodeficiency virus (HIV) will require knowledge of the immune responses that correlate with protection. During the acute phase of HIV infection the host immune responses appear to control viral replication. It is thought that virus-specific cellular immunity is intimately involved in this viral control. We have developed a model system to measure the entire T cell response and viral evolution in the face of this onslaught in rhesus macaques during the acute phase of infection with molecularly cloned simian immunodeficiency virus (SIV). We used intracellular cytokine staining (ICS) of peripheral blood mononuclear cells (PBMCs) from animals during the acute phase of viral infection stimulated with peptides spanning the entire protein sequence of SIV to determine which peptides were recognized by CD8 and CD4 positive T cells. Furthermore, we sequenced the entire virus during the acute phase of infection. This approach has proved highly effective for measuring acute phase T cell responses and viral evolution in SIV-infected rhesus macaques and might facilitate the definition of cellular immune responses in HIV-infected humans during the acute phase.

Vaccine safety. Analysis of oral polio vaccine CHAT stocks.

Batches of experimental oral vaccines against poliovirus (OPV CHAT) that were administered in Central Africa in the 1950s have been implicated in the origin of the AIDS pandemic because of possible retroviral contamination during the vaccine's manufacture, which allegedly involved chimpanzee kidney cells1. Here we use a molecular analysis to show that two CHAT type-1 polio vaccine stocks were prepared from macaque and not chimpanzee cells, and contain neither human nor simian immunodeficiency virus sequences. Our results do not support the hypothesis that these materials were responsible for the transmission of the AIDS virus to humans.

Protection of Macaca nemestrina from disease following pathogenic simian immunodeficiency virus (SIV) challenge: utilization of SIV nucleocapsid mutant DNA vaccines with and without an SIV protein boost.

Molecular clones were constructed that express nucleocapsid (NC) deletion mutant simian immunodeficiency viruses (SIVs) that are replication defective but capable of completing virtually all of the steps of a single viral infection cycle. These steps include production of particles that are viral RNA deficient yet contain a full complement of processed viral proteins. The mutant particles are ultrastructurally indistinguishable from wild-type virus. Similar to a live attenuated vaccine, this approach should allow immunological presentation of a full range of viral epitopes, without the safety risks of replicating virus. A total of 11 Macaca nemestrina macaques were inoculated with NC mutant SIV expressing DNA, intramuscularly (i.m.) in one study and i.m. and subcutaneously in another study. Six control animals received vector DNA lacking SIV sequences. Only modest and inconsistent humoral responses and no cellular immune responses were observed prior to challenge. Following intravenous challenge with 20 animal infectious doses of the pathogenic SIV(Mne) in a long-term study, all control animals became infected and three of four animals developed progressive SIV disease leading to death. All 11 NC mutant SIV DNA-immunized animals became infected following challenge but typically showed decreased initial peak plasma SIV RNA levels compared to those of control animals (P = 0.0007). In the long-term study, most of the immunized animals had low or undetectable postacute levels of plasma SIV RNA, and no CD4(+) T-cell depletion or clinical evidence of progressive disease, over more than 2 years of observation. Although a subset of immunized and control animals were boosted with SIV(Mne) proteins, no apparent protective benefit was observed. Immunization of macaques with DNA that codes for replication-defective but structurally complete virions appears to protect from or at least delay the onset of AIDS after infection with a pathogenic immunodeficiency virus. With further optimization, this may be a promising approach for vaccine development.

Variable constraints on the principal immunodominant domain of the transmembrane glycoprotein of human immunodeficiency virus type 1.

Lentiviruses have in their transmembrane glycoprotein (TM) a highly immunogenic structure referred to as the principal immunodominant domain (PID). The PID forms a loop of 5 to 7 amino acids between two conserved cysteines. Previous studies showed that envelope (Env) glycoprotein functions of feline immunodeficiency virus (FIV) could be retained after extensive mutation of the PID loop sequence, in spite of its high conservation. In order to compare Env function in different lentiviruses, either random mutations were introduced in the PID loop sequence of human immunodeficiency virus type 1 (HIV-1) or the entire HIV-1 PID loop was replaced by the corresponding PID loop of FIV or simian immunodeficiency virus (SIV). In the macrophage-tropic HIV-1 ADA Env, mutations impaired the processing of the gp160 Env precursor, thereby abolishing viral infectivity. However, 6 of the 108 random Env mutants that were screened retained the capacity to induce cell membrane fusion. The SIV and FIV sequences and five random mutations were then introduced in the context of T-cell-line-adapted HIV-1 LAI which, although phenotypically distant from HIV-1 ADA, has an identical PID loop sequence. In contrast to the situation for HIV-1 ADA mutants, the cleavage of the Env precursor was unaffected in most HIV-1 LAI mutants. Such mutations, however, resulted in increased shedding of the gp120 surface glycoprotein (SU) from the gp41 TM. The HIV-1 LAI Env mutants showed high fusogenic efficiency. Three Env mutants retained the capacity to mediate virus entry in target cells, although less efficiently than the wild-type Env, and allowed the reconstitution of infectious molecular clones. These results indicated that in HIV-1, like FIV, the conserved PID sequence can be changed without impairing Env function. However, functional constraints on the PID of HIV-1 vary depending on the structural context of Env, presumably in relation to the role of the PID in the interaction of the SU and TM subunits and the stability of the Env complex.

Toward a poliovirus-based simian immunodeficiency virus vaccine: correlation between genetic stability and immunogenicity.

Recombinant polioviruses expressing foreign antigens may provide a convenient vaccine vector to engender mucosal immunity. Replication-competent chimeric viruses can be constructed by fusing foreign antigenic sequences to several positions within the poliovirus polyprotein. Artificial cleavage sites ensure appropriate proteolytic processing of the recombinant polyprotein, yielding mature and functional viral proteins. To study the effect of the position of insertion, two different recombinant polioviruses were examined. A small amino-terminus insertion delayed virus maturation and produced a thermosensitive particle. In contrast, insertion at the junction between the P1 and P2 regions yielded a chimeric poliovirus that replicated like the wild type. Eight different chimeras were constructed by inserting simian immunodeficiency virus (SIV) sequences at the P1/P2 junction. All recombinant viruses replicated with near-wild-type efficiency in tissue culture cells and expressed high levels of the SIV antigens. One of the inserted fragments corresponding to gp41 envelope protein was N-glycosylated but was not secreted. Inserted sequences were only partially retained after few rounds of replication in HeLa cells. This problem could be remedied to some extent by altering the sequences flanking the insertion point. Reducing the homology of the direct repeats by 37% decrease the propensity of the recombinant viruses to delete the insert. To determine the immunogenic potential of the recombinants, mice susceptible to poliovirus infection were inoculated intraperitoneally. The antibody titers elicited against Gag p17 depended on the viral doses and the number of inoculations. In addition, recombinants which display higher genetic stability were more effective in inducing an immune response against the SIV antigens, and inoculation with a mix of recombinants carrying different SIV antigens (a cocktail of recombinants) elicited humoral responses against each of the individual SIV sequences.

Simian immunodeficiency virus needlestick accident in a laboratory worker

The macaque monkey infected with simian immunodeficiency virus (SIV) is an animal model of the acquired immunodeficiency syndrome. We investigated a laboratory worker who was exposed by needlestick accident to blood from an SIV-infected macaque. Seroreactivity to SIV developed within 3 months of exposure, with antibody titres peaking from the third to the fifth month and declining thereafter. Polymerase chain reaction for SIV sequences and cultures of peripheral-blood mononuclear cells failed to show infection. Inoculation of an SIV-negative monkey with blood from the worker did not cause infection. Animal-care and laboratory workers should adhere strictly to recommended procedures to avoid accidental exposures when working with SIV-infected animals or specimens.

Detection of unusual RNA folding regions in HIV and SIV sequences

We have developed a method for detecting more stable and significant folding regions relative to others in the sequence. The algorithm is based on the calculation of the lowest free energy of RNA secondary structures and Monte Carlo simulation. For any given RNA segment, the stability and statistical significance of RNA folding are assessed by two measures: the stability score and the significance score. The stability score measures the degree of thermodynamic stability of the segment between all possible biological segments in the RNA sequence. The significance score characterizes the specific arrangement of the nucleotides in the segment that could imply a structural role for the sequence information. Using these two measures, we are able to detect a series of distinct folding regions where highly stable and statistically significant secondary structures occur in human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) sequences.

Genetic Analysis and Infection of SIVAGM and SIVMND

Recently, the authors determined the partial sequence of simian immunodeficiency virus (SIV) from the mandrill (SIVMND) and found SIVMND to be a new member of the HIV/SIV group, equidistant from other members, including SIVAGM. Experimentally, the African green monkey and cynomolgus monkey could be infected with SIVAGM and the cottontop tamarin with SIVMND. However, no clinical sign of an AIDS-like disease was observed in these monkeys.